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Plant Endo-β-mannanase Activity Assay
植物内型(endo)-β-甘露聚糖酶活性试验   

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参见作者原研究论文

本实验方案简略版
The Plant Journal
May 2013

Abstract

Endo-β–mannanases in plant require post-translational modification, such as N-glycosylation and disulfide-linked dimerization, for their catalytic activity. Determination of the plant endo-β–mannanase activity needs to modify the assay conditions for optimizing their enzymatic reaction. Here, we describe a modified method for plant endo-β–mannanase assay. A high-salt buffer without thiol reductants is required for effective extraction of the enzyme. The enzyme is able to digest water-insoluble AZCL galactomannan to release water soluble dyed fragments, which is detected through measurement of absorbance at 590 nm wavelength. Increase in absorbance at 590 nm is correlated directly with enzyme activity.

Keywords: Endo-ß-mannanse (内ß-甘露聚糖酶), Poplar (杨树), AZC L-galactomannan (l-galactomannan AZC), PtrMAN6 (ptrman6)

Materials and Reagents

  1. Liquid nitrogen
  2. BCA Reagent (Tiangen Biotech, catalog number: PA115-01 )
  3. Bovine serum albumin
  4. AZC L-galactomannan (Megazyme, catalog number: I-AZGMA )
  5. Commercial Aspergillus niger endo-β-mannanase (Megazyme, catalog number: E-BMANN )
  6. 100 mM phenylmethanesulfonyl fluoride (PMSF) (see Recipes)
  7. 0.5 M ethylene diaminete traacetic acid (EDTA) (see Recipes)
  8. Extraction buffer (see Recipes)
  9. 0.1 M sodium acetate buffer (pH 5.0) (see Recipes)

Equipment

  1. Mortar and pestle
  2. 10,000 Mr cut-off filter (EMD Millipore, catalog number: UFC801096 )
  3. Centrifuge
  4. Incubator shaker
  5. Water bath
  6. Microplate reader or Spectrophotometer

Procedure

  1. Samples (~ 10 g developing xylem or leaves from one-year-old poplar) are ground in liquid nitrogen to a fine powder and homogenized at 4 °C in 1.5-volume fold of extraction buffer for 1 h (strong enzymatic activity can be detected in developing xylem).
  2. The homogenate is centrifuged at 10,000 x g for 30 min at 4 °C.
  3. The supernatant is then passed through a 10,000 Mr cut-off filter and dehydrated to < 500 μl, then the protein is diluted to ~1 μg/μl in 0.1 M sodium acetate buffer (pH 5.0) at 4 °C.
  4. The protein extraction is measured by BCA Reagent using bovine serum albumin as a standard.
  5. 200 μl of reaction mixture containing 100 μl of 1% AZC L-galactomannan (w/v, in 0.1 M sodium acetate buffer, pH 5.0) and 20 μg of extracted proteins or heated inactive proteins (100 °C 10 min, as control) is incubated at 40 °C for 2 h with continuous shake.
  6. The reaction mixture is boiled at 100 °C for 5 min and centrifuged at 12,000 x g for 5 min.
  7. The absorbance (A) of the supernatant at 590 nm is determined. The background values (A0) obtained using heated inactive proteins are subtracted from values (A1) obtained using active extract (A = A1 - A0).
  8. Standardization
    1. Enzyme activity of a serial dilutions of a commercial Aspergillus niger endo-β-mannanase (E-BMANN, Megazyme) is determined under the conditions: 200 μl of reaction mixture containing 100 μl of 1% AZC L-galactomannan and E-BMANN is incubated at 40 °C for 2 h.
    2. A standard curve correlated with E-BMANN activity is shown in Figure 1. For absorbance values in a range of 0.05~0.9, these values can be calculated by reference to the equation: Y = SX + C. Where:
      Y = endo-β-Mannanase activity (in micro-Units/assay, i.e. per 200 μl)
      S = Slope of the calibration graph
      X = Absorbance of the reaction at 590 nm (A)
      C = Intercept on the Y-axis
    3. According to the manufacturer’s instruction, one Unit of activity is defined as the amount of enzyme required to release one micromole of mannose reducing-sugar equivalents per minute under the defined assay conditions (1 micro-Unit = 1 pmol/min).
  9. Calculation of enzyme activity: endo-β-Mannanase activity in the sample is determined by reference to the standard curve to convert absorbance values to micro-Units per assay (Y), then further to micro-Units per μg protein (Y/[20 μg protein], pmol/min/[μg protein]).


    Figure 1. Endo-β-mannanase standard curve on the commercial endo-β-mannanase.

Recipes

  1. 100 mM Phenylmethanesulfonyl fluoride (PMSF) 10 ml
    Mix 0.174 g of PMSF with 10 ml isopropanol
    Store in small aliquots at -20 °C
  2. 0.5 M Ethylene diamine tetraacetic acid (EDTA) (pH 8.0) (1 L)
    Dissolve 186.1 g EDTA-Na.2H2O in 800 ml dH2O
    Adjust pH to 8.0 with NaOH (~20 g NaOH particles)
    Add dH2O to 1 L
    Autoclave at 121 °C for 20 min
    Store at RT
  3. Extraction buffer (1 L)
    1 M sodium acetate buffer (pH 5.0)
    10 mM EDTA
    10 mM sodium azide
    3 mM PMSF
    Mix 57 ml glacial acetic acid (1.05 g/ml) and 20 ml 0.5 M EDTA with 800 ml dH2O
    Adjust pH to 5.0 with NaOH
    Add 0.65 g sodium azide
    Add dH2O to 1 L
    Add 30 μl of 100 mM PMSF per ml extraction buffer immediately before use
    Note: Do not add the sodium azide until pH is adjusted. Acidification of sodium azide will release a poisonous gas.
  4. 0.1 M sodium acetate buffer (pH 5.0)
    Mix 5.7 ml glacial acetic acid (1.05 g/ml) with 800 ml dH2O
    Adjust pH to 5.0 with NaOH
    Add 0.65 g sodium azide
    Add dH2O to 1 L

Acknowledgments

This protocol was adapted from Zhao et al. (2013).

References

  1. Zhao, Y., Song, D., Sun, J. and Li, L. (2013). Populus endo-beta-mannanase PtrMAN6 plays a role in coordinating cell wall remodeling with suppression of secondary wall thickening through generation of oligosaccharide signals. Plant J 74(3): 473-485.

简介

对于其催化活性,植物中的内切-β-甘露聚糖酶需要翻译后修饰,例如N-糖基化和二硫键连接的二聚化。 植物内切-β-甘露聚糖酶活性的测定需要改变测定条件以优化它们的酶反应。 在这里,我们描述了植物内切-β-甘露聚糖酶测定的修改方法。 为了有效地提取酶,需要没有硫醇还原剂的高盐缓冲液。 该酶能够消化水不溶性AZCL半乳甘露聚糖以释放水溶性染色片段,其通过测量在590nm波长处的吸光度来检测。 在590nm处的吸光度的增加与酶活性直接相关

关键字:内ß-甘露聚糖酶, 杨树, l-galactomannan AZC, ptrman6

材料和试剂

  1. 液氮
  2. BCA试剂(Tiangen Biotech,目录号:PA115-01)
  3. 牛血清白蛋白
  4. AZC L-半乳甘露聚糖(Megazyme,目录号:I-AZGMA)
  5. 商业黑曲霉内切-β-甘露聚糖酶(Megazyme,目录号:E-BMANN)
  6. 100mM苯甲磺酰氟(PMSF)(参见配方)
  7. 0.5M乙二胺四乙酸(EDTA)(参见配方)
  8. 提取缓冲液(参见配方)
  9. 0.1 M乙酸钠缓冲液(pH 5.0)(参见配方)

设备

  1. 砂浆和杵
  2. 10,000 截止过滤器(EMD Millipore,目录号:UFC801096)
  3. 离心机
  4. 培养箱振动器
  5. 水浴
  6. 酶标仪或分光光度计

程序

  1. 将样品(约10g产生的木质部或来自一年生杨树的叶)在液氮中研磨成细粉并在1.5体积倍数的提取缓冲液中在4℃下均化1小时(可以检测到强的酶活性在开发木质部)。
  2. 匀浆在10,000xg在4℃离心30分钟。
  3. 然后使上清液通过10,000μm截止滤器,并脱水, 500μl,然后在4℃下在0.1M乙酸钠缓冲液(pH5.0)中将蛋白质稀释至〜1μg/μl。
  4. 使用牛血清白蛋白作为标准通过BCA试剂测量蛋白质提取。
  5. 将含有100μl1%AZC L-半乳甘露聚糖(w/v,在0.1M乙酸钠缓冲液中,pH5.0)和20μg提取的蛋白质或加热的无活性蛋白质(100℃10分钟,作为对照)的200μl反应混合物,在40℃下连续振荡孵育2小时
  6. 将反应混合物在100℃下煮沸5分钟,并以12,000×g离心5分钟。
  7. 测定上清液在590nm处的吸光度(A)。使用加热的无活性蛋白获得的背景值(A sub)从使用活性提取物(A = A 1)获得的值(A sub) - A 0 )。
  8. 标准化
    1. 在以下条件下测定市售黑曲霉内切-β-甘露聚糖酶(E-BMANN,Megazyme)的系列稀释液的酶活性:将200μl含有100μl1%AZC L-半乳甘露聚糖和E-BMANN在40℃温育2小时。
    2. 与E-BMANN活性相关的标准曲线如图1所示。对于0.05〜0.9范围内的吸光度值,可以参考下列公式计算这些值:Y = SX + C其中:
      Y =内切-β-甘露聚糖酶活性(以微单位/测定,即每200μl为)
      S =校准曲线的斜率
      X =在590nm处的反应吸光度(A)
      C =在Y轴上截取
    3. 根据制造商的说明,一个活性单位定义为在限定的测定条件(1微单位= 1pmol/min)下每分钟释放1微摩尔甘露糖还原糖当量所需的酶量。
  9. 酶活性的计算:样品中内切-β-甘露聚糖酶活性通过参照标准曲线确定,以将吸光度值转化为每个测定的微单位(Y),然后进一步改变为每μg蛋白的微单位(Y/[20 μg蛋白质],pmol/min/[μg蛋白质])

    图1.商业内切-β-甘露聚糖酶上的内切-β-甘露聚糖酶标准曲线。

食谱

  1. 100mM苯基甲磺酰氟(PMSF)10ml 将0.174g PMSF与10ml异丙醇混合 以小等分试样储存在-20℃下
  2. 0.5M乙二胺四乙酸(EDTA)(pH8.0)(1L) 在800ml dH 2 O中溶解186.1g EDTA-Na 2 H 2 2H 2 O。 用NaOH(〜20g NaOH颗粒)将pH调节至8.0 将dH <2> O添加到1 L
    在121℃高压灭菌20分钟
    在RT存储
  3. 提取缓冲液(1 L)
    1M乙酸钠缓冲液(pH5.0) 10 mM EDTA
    10mM叠氮化钠 3 mM PMSF
    将57ml冰醋酸(1.05g/ml)和20ml 0.5M EDTA与800ml dH 2 O混合。
    用NaOH调节pH至5.0 加入0.65g叠氮化钠 将dH <2> O添加到1 L
    在使用前立即加入30μl100mM PMSF/ml提取缓冲液
    注意:在调整pH之前,不要添加叠氮化钠。 叠氮化钠的酸化将释放有毒气体。
  4. 0.1M乙酸钠缓冲液(pH5.0) 将含有800ml dH 2 O的5.7ml冰醋酸(1.05g/ml)混合
    用NaOH调节pH至5.0 加入0.65g叠氮化钠 将dH <2> O添加到1 L

致谢

该协议改编自Zhao等人(2013)。

参考文献

  1. Zhao,Y.,Song,D.,Sun,J.and Li,L。(2013)。 山杨中β-甘露聚糖酶PtrMAN6在协调细胞壁重塑和抑制次生壁增厚中起作用 通过产生寡糖信号。 Plant J 74(3):473-485。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhao, Y. and Li, L. (2013). Plant Endo-β-mannanase Activity Assay. Bio-protocol 3(17): e883. DOI: 10.21769/BioProtoc.883.
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